1. Field of the Invention
The present invention is directed to a method for forming surface-enhanced Raman scattering (SERS) substrates. In particular, it is directed to a method for forming SERS substrates using nanoparticle-based films.
2. Description of the Related Technology
Raman scattering spectroscopy (RSS) is the measurement of the wavelength and intensity of light scattered inelastically from a molecule. Raman scattering is the result of an inelastic collision of a photon with atoms. Both elastic and inelastic collisions occur when light interacts with a molecule. In elastic collisions (Rayleigh scattering), an atom is excited from a ground state to a higher energy state and then relaxes back to the original ground state, thereby emitting a photon at the same frequency as the incident light. However in an inelastic collision, the excited molecule relaxes to a different vibrational state rather than the original state, thereby scattering energy different from that of the incident light. If the scattered energy is higher than the incident light it is called an Anti-Stokes line (blue shifted), if it is lower it is called a Stokes line (red shifted).
FIG. 1 represents a schematic of the Raman spectrum. RS gives information about the characteristic vibrational states of the chemical bonds of molecules. It is a widely used spectroscopic tool for the determination of molecular structure and for compound identification. Despite all these advantages, its use has been somewhat limited due to its poor efficiency. RSS when compared to fluorescence spectroscopy has a small Scattering Cross Section (10−30 cm2 per molecule when compared to 10−16 cm2 for fluorescence) thus reducing the possibility of analyzing compounds of biological significance due to the generally low concentration of analytes in biological samples of this size.
However, due to the availability of better SERS substrates in the past decade, the SERS technique has seen a remarkable surge in its use, especially for the detection of biologically significant molecules such as toxins and disease-related molecules.
There is a way to greatly enhance the Raman signal by using specially structured metallic substrates, typically constructed of Ag, Au, and Cu. Such Surface Enhanced Raman Scattering was first reported in 1974 where a large enhancement of Raman signals of pyridine molecules adsorbed on electrochemically roughened silver electrodes was observed. SERS amplification factors of between 106˜1016 have been achieved using a wide range of SERS substrates. This enhancement effect has made RS an increasingly important analytical tool in biological sciences. It must be mentioned though that despite numerous physical models that have been proposed to explain SERS, it is generally agreed that a complete theoretical understanding of the SERS mechanisms remains elusive.
There is a serious drawback that plagues all SERS substrates and makes them less than promising candidates for SERS imaging. Typical SERS substrates are fabricated by methods that result in the noble metal nanostructures stochastically distributed over the substrate surface, e.g. electrochemically roughened electrodes, sputtered films, chemically etched films, electroless deposited films, and colloidal metal particles. Another way to obtain large amplification in Raman scattering is to place the sample in close proximity to a sharp metallic tip. This stochastic nature leads to randomly distributed Raman “hot spots” and results in a lack of reproducibility in most SERS substrates. It is therefore desirable to create a substrate that can induce enhanced Raman scattering homogeneously over the entire sample area in order to provide enhanced Raman imaging.
By using controlled patterning of the nanostructures with electron-beam and other lithographic techniques, little randomness remains and one can expect SERS to be homogeneous across the proposed substrate. Such substrates are now commercially available (Mesophotonics Limited, Southampton, UK). Fabrication of such substrates involves a multi-step process and the resulting substrates are quite expensive. Such substrates are also small in size, for example, between 4 mm×4 mm.
An advantage of RS is that it offers, without the need for labeling, molecular-level specificity at sub-micron spatial resolutions. While a number of other imaging techniques employing confocal microscopes also offer molecular-level specificity at sub-micron resolutions, all of them require certain forms of labeling on the sample. These various labeling techniques, e.g. Green Fluorescence Protein, immuno-labeling, staining, fluorescence, luminescence, etc, inevitably alter the very sample under examination as they achieve the specificity critical for bio-imaging.
RS, on the other hand, probes the inherent vibrational states of a molecule and therefore attains chemical specificity without the need for labeling. However, the Raman scattering signals from those vibrational states are relatively weak. In order to obtain a satisfactory signal-to-noise ratio, one has to either increase the intensity of the probing laser or resort to surface enhanced Raman scattering. For biological applications, increased laser intensity often limits the in vivo imaging capability of a system. It is therefore desirable to create SERS structures that permit the ability to use enhanced bio-imaging.
One of the most common methods to enhance the Raman signal is by using colloidal silver or gold nanoparticles. The largest SERS signal amplification is achieved when the analyte molecule is sandwiched between two nanoparticles and when the polarization vector, i.e. the direction of the oscillating E-field of the laser's electromagnetic field, is along the line connecting the centers of the Ag nanoparticles. Amplification factors in the range of 6*106 to 2.5*1010 have been predicted when the separation between two Ag nanoparticles of diameter 90 nm is varied between 5.5 and 1.0 nm. When the polarization vector is perpendicular to the Ag nanoparticles, the maximum amplification factor is relatively small (about 1 to 10). In FIG. 2 the polarization vector with respect to the molecules is shown The amplification factor for the geometry indicated in [c] is intermediate between cases [a] and [b] See e.g. H. Xu, et al. “Spectroscopy of single Hemoglobin molecules by surface enhanced Raman scattering,” Phys. Rev. Lett. 83, 43574360 (1999).
To produce such favorable geometries as depicted in FIG. 2, and in other compact aggregates, the analyte is incubated in an Ag colloidal suspension (in water or other suitable liquid organic carrier) with 1.0-10.0 mM NaCl solution. The role of NaCl is as an aggregating agent. The Ag aggregates are then sorted according to their size and compact aggregates (two to about ten particles each) and isolated for further study using SERS. There are two drawbacks to this technique of creating compact Ag aggregates. First, there is poor control over the size of the aggregates produced. The creation of hot spots for Raman scattering largely results as accidental byproducts of the technique. Second, the yield of the desired aggregates is very low and the suitable portions of the nanoparticle array on the substrate must then be selected from a mixture of larger aggregates before they can be used for SERS study. This prevents the fabrication of suitable SERS substrates on a large scale.
U.S. Pat. No. 6,989,897 B2 provides metal-coated nanocrystalline silicon as an active surface-enhanced Raman scattering substrate. This patent provides the required spacing between active nanoparticles by patterning the substrate to receive the nanoparticles at desired locations and then locating the nanoparticles in the patterned locations on the substrate.
U.S. Pat. No. 6,242,264 provides a dip coating method for coating nanoparticles onto a surface of a glass slide for use in Raman scattering. In the process, the glass slide is dipped first coated with an adhesion promoting material and then dipped into a solution containing the nanoparticles for a time period sufficient to provide the required density of nanoparticles on the surface of the glass slide. This method does not appear to provide fractal aggregates of the nanoparticles but instead generates concentration gradients along the slide due to the use of the dip coating method. Further, dip coating is limited in its commercial applicability.
U.S. patent application publication No. 2006/0060885 discloses a method for depositing a conductive nanoparticle layer onto a substrate surface. A solution of stabilized nanoparticles is applied to a substrate surface and heated to remove liquid vehicle and stabilizer until a nanoparticle layer is formed. Heating is continued until a minimum conductivity of 1 Siemens/centimeter is achieved in order to provide the conductive coating.
A fractal is a self-similar geometrical object—it looks the same at any length scale. In practice this self similarity does not extend infinitely due to the finite sample size. Jean-Francois Gouyet, Physics and Fractal Structure, Springer, Berlin, 1996, Chapter 1.
Metallic fractal aggregates can exhibit some of the highest SERS signal amplification factors. V. M. Shalaev, Nonlinear Optics of Random Media. Fractal Composites and Metal-Dielectric Films, Springer, Heidelberg (2000). Ideally, a fractal has the property that it looks the same at any scale. In practice this structural scale invariance will be limited by finite sample size.
Fractal aggregates of metallic colloidal particles can provide enhancement for various linear and nonlinear optical responses, including Raman scattering. The basic mechanism that gives rise to such enhancement arises from the localization of optical plasmon excitations within small parts (“hot-spots”) of a fractal aggregate. Such “hot spots”, are usually much smaller (tens of nm) than the size of the fractal and often much smaller than the wavelength. Fractal structures, unlike translationally invariant media, cannot support propagating waves and hence can confine electromagnetic field to very small regions of the substrate. If sufficiently concentrated, the enhanced electromagnetic fields in the hot spots can result in SERS signal amplification. The small areas, where the fractal optical excitations are localized, have very different local structures and, therefore, are characterized by different resonant frequencies.
The various nano-scale regions, where the resonant fractal excitations are localized, act as a collection of different optical “nano-resonators”, resulting in a distribution of resonance frequencies in the visible and IR spectral ranges and can have resonance quality-factors as large as 103. When Stokes shifts are small, the SERS signal is roughly proportional to the local field raised to the fourth power and, therefore, it can be enhanced up to 1012 in the fractal hot spots. Such amplification factors can be further enhanced by chemical enhancement. See V. M. Shalaev, Nonlinear Optics of Random Media: Fractal Composites and Metal-Dielectric Films, Springer, Heidelberg (2000).
Therefore, there is a need in the field to provide a method for forming SERS substrates in manner that permits control of the average distance between nanoparticles and to be able to produce SERS substrates in larger numbers at lower cost.